Class II cytokine receptor-7

ABSTRACT

Novel receptor polypeptides, polynucleotides encoding the polypeptides, and related compositions and methods are disclosed. The polypeptides comprise an extracellular domain of a cell-surface receptor that is expressed in kidneys, pancreas, prostate, adrenal cortex and nervous tissue. The polypeptides may be used within methods for detecting ligands that promote the proliferation and/or differentiation of these organs.

The present application is a divisional of U.S. patent application Ser.No. 08/943,087, now U.S. Pat. No. 5,945,511, which is acontinuation-in-part of U.S. patent application Ser. No. 08/803,305filed Feb. 20, 1997, which is now abandoned.

BACKGROUND OF THE INVENTION

Cytokines are soluble proteins that influence the growth anddifferentiation of many cell types. Their receptors are composed of oneor more integral membrane proteins that bind the cytokine with highaffinity and transduce this binding event to the cell through thecytoplasmic portions of the certain receptor subunits. Cytokinereceptors have been grouped into several classes on the basis ofsimilarities in their extracellular ligand binding domains. For example,the receptor chains responsible for binding and/or transducing theeffect of interferons (IFNs) are members of the type II cytokinereceptor family (CRF2), based upon a characteristic 200 residueextracellular domain. The demonstrated in vivo activities of theseinterferons illustrate the enormous clinical potential of, and need for,other cytokines, cytokine agonists, and cytokine antagonists.

SUMMARY OF THE INVENTION

The present invention fills this need by providing novel cytokinereceptors and related compositions and methods. In particular, thepresent invention provides for an extracellular ligand-binding region ofa mammalian Zcytor7 receptor, alternatively also containing either atransmembrane domain or both an intracellular domain and a transmembranedomain.

Within one aspect, the present invention provides an isolatedpolynucleotide encoding a ligand-binding receptor polypeptide. Thepolypeptide comprises a sequence of amino acids selected from the groupconsisting of (a) residues 30 through 250 of SEQ ID NO:2; (b) allelicvariants of (a); and (c) sequences that are at least 80% identical to(a) or (b). Within one embodiment, the polypeptide comprises residues 30through 250 of SEQ ID NO:2. Within another embodiment, the polypeptideencoded by the isolated polynucleotide further comprises a transmembranedomain. The transmembrane domain may comprise residues 251 through 274of SEQ ID NO:2, or an allelic variant thereof. Within anotherembodiment, the polypeptide encoded by the isolated polynucleotidefurther comprises an intracellular domain, such as an intracellulardomain comprising residues 275 through 553 of SEQ ID NO:2, or an allelicvariant thereof. Within further embodiments, the polynucleotide encodesa polypeptide that comprises residues 1 through 553, 1 through 274, 1through 250, 30 through 274 or 30 through 553 of SEQ ID NO:2. Within anadditional embodiment, the polypeptide further comprises an affinitytag. Within a further embodiment, the polynucleotide is DNA. Alsoclaimed are the isolated polypeptides encoded by these polynucleotides.

Within a second aspect of the invention there is provided an expressionvector comprising (a) a transcription promoter; (b) a DNA segmentencoding a ligand-binding receptor polypeptide, wherein theligand-binding receptor polypeptide comprises a sequence of amino acidsselected from the group consisting of: (i) residues 30 through 250 ofSEQ ID NO:2; (ii) allelic variants of (i); and (iii) sequences that areat least 80% identical to (i) or (ii); and (c) a transcriptionterminator, wherein the promoter, DNA segment, and terminator areoperably linked. The ligand-binding receptor polypeptide may furthercomprise a secretory peptide, a transmembrane domain, a transmembranedomain and an intracellular domain, or a secretory peptide, atransmembrane domain and an intracellular domain.

Within a third aspect of the invention there is provided a culturedeukaryotic cell into which has been introduced an expression vector asdisclosed above, wherein said cell expresses a receptor polypeptideencoded by the DNA segment. Within one embodiment, the cell furtherexpresses a necessary receptor subunit which forms a functional receptorcomplex. Within another embodiment, the cell is dependent upon anexogenously supplied hematopoietic growth factor for proliferation.

Within a fourth aspect of the invention there is provided an isolatedpolypeptide comprising a sequence selected from the group consisting of(a) residues 30, a valine, through residue 250, a lysine, of SEQ IDNO:2; (b) allelic variants of (a); and (c) sequences that are at least80% identical to (a) or (b), wherein said polypeptide is substantiallyfree of transmembrane and intracellular domains ordinarily associatedwith hematopoietic receptors. Also claimed are polypeptides comprised ofa sequence defined by residues 30, a valine, through residue 274, atyrosine; and a polypeptide comprised of a sequence defined by residues30, a valine, through residue 553 an asparagine. Also claimed are thepolypeptides and polynucleotides defined by the sequences of SEQ ID NOs:13-60.

Within a further aspect of the invention there is provided a chimericpolypeptide consisting essentially of a first portion and a secondportion joined by a peptide bond. The first portion of the chimericpolypeptide consists essentially of a ligand binding domain of areceptor polypeptide selected from the group consisting of (a) areceptor polypeptide as shown in SEQ ID NO:2; (b) allelic variants ofSEQ ID NO:2; and (c) receptor polypeptides that are at least 80%identical to (a) or (b). The second portion of the chimeric polypeptideconsists essentially of an affinity tag. Within one embodiment theaffinity tag is an immunoglobulin F_(c) polypeptide. The invention alsoprovides expression vectors encoding the chimeric polypeptides and hostcells transfected to produce the chimeric polypeptides.

The present invention also provides for an isolated polynucleotideencoding a polypeptide selected from a group defined SEQ ID NO:2consisting of residues 1 through 250, residues 1 through 274, residues 1through 553, residues 2 through 250, residues 2 through 274, residues 2through 553, residues 251 through 274, residues 251 through 553 andresidues 275 through 553. Also claimed are the isolated polypeptideexpressed by these polynucleotides.

The invention also provides a method for detecting a ligand within atest sample, comprising contacting a test sample with a polypeptide asdisclosed above, and detecting binding of the polypeptide to ligand inthe sample. Within one embodiment the polypeptide further comprisestransmembrane and intracellular domains. The polypeptide can be membranebound within a cultured cell, wherein the detecting step comprisesmeasuring a biological response in the cultured cell. Within anotherembodiment, the polypeptide is immobilized on a solid support.

Within an additional aspect of the invention there is provided anantibody that specifically binds to a polypeptide as disclosed above, aswell as an anti-idiotypic antibody which binds to the antigen-bindingregion of an antibody to Zcytor7.

In still another aspect of the present invention, polynucleotide primersand probes are provided which can detect mutations in the Zcytor7 gene.The polynucleotide probe should at least be 20-25 bases in length,preferably at least 50 bases in length and most preferably about 80 to100 bases in length. In addition to the detection of mutations, theseprobes can be used to discover the Zcytor7 gene in other mammalianspecies. The probes can either be positive strand or anti-sense strands,and they can be comprised of DNA or RNA.

These and other aspects of the invention will become evident uponreference to the following detailed description and the attacheddrawing.

DETAILED DESCRIPTION OF THE INVENTION

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems.

“Operably linked”, when referring to DNA segments, indicates that thesegments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in the promoter andproceeds through the coding segment to the terminator.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

The term “receptor” is used herein to denote a cell-associated protein,or a polypeptide subunit of such a protein, that binds to a bioactivemolecule (the “ligand”) and mediates the effect of the ligand on thecell. Binding of ligand to receptor results in a conformational changein the receptor (and, in some cases, receptor multimerization, i.e.,association of identical or different receptor subunits) that causesinteractions between the effector domain(s) and other molecule(s) in thecell. These interactions in turn lead to alterations in the metabolismof the cell. Metabolic events that are linked to receptor-ligandinteractions include gene transcription, phosphorylation,dephosphorylation, cell proliferation, increases in cyclic AMPproduction, mobilization of cellular calcium, mobilization of membranelipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis ofphospholipids. The term “receptor polypeptide” is used to denotecomplete receptor polypeptide chains and portions thereof, includingisolated functional domains (e.g., ligand-binding domains).

A “secretory signal sequence” is a DNA sequence that encodes apolypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

A “soluble receptor” is a receptor polypeptide that is not bound to acell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains.Soluble receptors can comprise additional amino acid residues, such asaffinity tags that provide for purification of the polypeptide orprovide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis or translated from alternatively spliced mRNAs. Receptorpolypeptides are said to be substantially free of transmembrane andintracellular polypeptide segments when they lack sufficient portions ofthese segments to provide membrane anchoring or signal transduction,respectively.

The present invention is based in part upon the discovery of a novel DNAsequence that encodes a protein having the structure of a cytokinereceptor, including the conserved WSXWS motif (SEQ ID NO: 10). Analysisof the tissue distribution of the mRNA corresponding to this novel DNAshowed that mRNA level was highest in pancreas, prostate, kidney andadrenal cortex followed by lower levels in testis, stomach, adrenalmedulla and thymus. The receptor has been designated “Zcytor7”.

Cytokine receptors subunits are characterized by a multi-domainstructure comprising a ligand-binding domain and an effector domain thatis typically involved in signal transduction. Multimeric cytokinereceptors include homodimers (e.g., PDGF receptor αα and ββ isoforms,erythropoietin receptor, MPL [thrombopoietin receptor], and G-CSFreceptor), heterodimers whose subunits each have ligand-binding andeffector domains (e.g., PDGF receptor αβ isoform), and multimers havingcomponent subunits with disparate functions (e.g., IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, and GM-CSF receptors). Some receptor subunits arecommon to a plurality of receptors. For example, the AIC2B subunit,which cannot bind ligand on its own but includes an intracellular signaltransduction domain, is a component of IL-3 and GM-CSF receptors. Manycytokine receptors can be placed into one of four related families onthe basis of their structures and functions. Class I hematopoieticreceptors, for example, are characterized by the presence of a domaincontaining conserved cysteine residues and the WSXWS motif (SEQ ID NO:10). Additional domains, including protein kinase domains; fibronectintype III domains; and immunoglobulin domains, which are characterized bydisulfide-bonded loops, are present in certain hematopoietic receptors.Cytokine receptor structure has been reviewed by Urdal, Ann. ReportsMed. Chem. 26:221-228, 1991 and Cosman, Cytokine 5:95-106, 1993. It isgenerally believed that under selective pressure for organisms toacquire new biological functions, new receptor family members arose fromduplication of existing receptor genes leading to the existence ofmulti-gene families. Family members thus contain vestiges of theancestral gene, and these characteristic features can be exploited inthe isolation and identification of additional family members.

Cell-surface cytokine receptors are further characterized by thepresence of additional domains. These receptors are anchored in the cellmembrane by a transmembrane domain characterized by a sequence ofhydrophobic amino acid residues (typically about 21-25 residues), whichis commonly flanked by positively charged residues (Lys or Arg). On theopposite end of the protein from the extracellular domain and separatedfrom it by the transmembrane domain is an intracellular domain.

The novel receptor of the present invention, Zcytor7, is a class IIcytokine receptor. These receptors usually bind to four-helix-bundlecytokines. Interleukin-10 and the interferons have receptors in thisclass (e.g., interferon-gamma alpha and beta chains and theinterferon-alpha/beta receptor alpha and beta chains). Class II cytokinereceptors are characterized by the presence of one or more cytokinereceptor modules (CRM) in their extracellular domains. The CRMs of classII cytokine receptors are somewhat different than the better known CRMsof class I cytokine receptors. While the class II CRMs contain twotype-III fibronectin-like domains, they differ in organization. Inparticular, they contain two WSXWS (SEQ ID NO: 10) motifs, one in eachfibronectin III-like domain. These WSXWS (SEQ ID NO: 10) motifs,however, are less conserved than those found in class I CRMs.

Zcytor7, like all known class II receptors except interferon-alpha/betareceptor alpha chain, has only a single class II CRM in itsextracellular domain. Zcytor7 appears to be a receptor for a helicalcytokine of the interferon/IL-10 class. Using the Zcytor7 receptor wecan identify ligands and additional compounds which would be ofsignificant therapeutic value. Furthermore, the extracellular portion ofZcytor7 extending from residue 30, a valine, through residue 250 of SEQID NO: 2 can be expressed and used as a soluble receptor todown-regulate the effects of the ligand of Zcytor7.

As was stated above, Zcytor7 was initially identified by the overallhomology to CRF2-4, an orphan Class II cytokine receptor. See LutfallaG. et al. Genomics, 16: 366-373 (1993). Analysis of a human cDNA cloneencoding Zcytor7 (SEQ ID NO: 1) revealed an open reading frame encoding553 amino acids (SEQ ID NO:2) comprising an extracellular ligand-bindingdomain of approximately 221 amino acid residues (residues 30-250 of SEQID NO:2), a transmembrane domain of approximately 24 amino acid residues(residues 251-274 of SEQ ID NO:2), and an intracellular domain ofapproximately 279 amino acid residues (residues 275-553 of SEQ ID NO:2).Those skilled in the art will recognize that these domain boundaries areapproximate and are based on alignments with known proteins andpredictions of protein folding. Deletion of residues from the ends ofthe domains is possible.

Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO: 1or a sequence complementary thereto, under stringent conditions. Ingeneral, stringent conditions are selected to be about 5° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Typical stringent conditions are those in whichthe salt concentration is at least about 0.02 M at pH 7 and thetemperature is at least about 60° C. As previously noted, the isolatedpolynucleotides of the present invention include DNA and RNA. Methodsfor isolating DNA and RNA are well known in the art. It is generallypreferred to isolate RNA from pancreas or prostate tissues although cDNAcan also be prepared using RNA from other tissues or isolated as genomicDNA. Total RNA can be prepared using guanidine HCl extraction followedby isolation by centrifugation in a CsCl gradient (Chirgwin et al.,Biochemistry 18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNAusing the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA69:1408-1412, 1972). Complementary DNA (cDNA) is prepared from poly(A)⁺RNA using known methods. Polynucleotides encoding ZCytor7 polypeptidesare then identified and isolated by, for example, hybridization or PCR.

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NOS:1 and 2 represent single alleles of the human ZCytor7receptor. Allelic variants of these sequences can be cloned by probingcDNA or genomic libraries from different individuals according tostandard procedures. Other specific embodiments include thepolynucleotides and polypeptides defined by SEQ ID NOs: 13-60.

The present invention further provides counterpart receptors andpolynucleotides from other species (“species orthologs”). Of particularinterest are ZCytor7 receptors from other mammalian species, includingmurine, porcine, ovine, bovine, canine, feline, equine, and otherprimate receptors. Species orthologs of the human ZCytor7 receptor canbe cloned using information and compositions provided by the presentinvention in combination with conventional cloning techniques. Forexample, a cDNA can be cloned using mRNA obtained from a tissue or celltype that expresses the receptor. Suitable sources of mRNA can beidentified by probing Northern blots with probes designed from thesequences disclosed herein. A library is then prepared from mRNA of apositive tissue or cell line. A receptor-encoding cDNA can then beisolated by a variety of methods, such as by probing with a complete orpartial human cDNA or with one or more sets of degenerate probes basedon the disclosed sequences. A cDNA can also be cloned using thepolymerase chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202),using primers designed from the sequences disclosed herein. Within anadditional method, the cDNA library can be used to transform ortransfect host cells, and expression of the cDNA of interest can bedetected with an antibody to the receptor. Similar techniques can alsobe applied to the isolation of genomic clones.

The present invention also provides isolated receptor polypeptides thatare substantially homologous to the receptor polypeptide of SEQ ID NO:2. By “isolated” is meant a protein or polypeptide that is found in acondition other than its native environment, such as apart from bloodand animal tissue. In a preferred form, the isolated polypeptide issubstantially free of other polypeptides, particularly otherpolypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. The term “substantially homologous” isused herein to denote polypeptides having 50%, preferably 60%, morepreferably at least 80%, sequence identity to the sequences shown in SEQID NO:2. Such polypeptides will more preferably be at least 90%identical, and most preferably 95% or more identical to SEQ ID NO:2.Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992.Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “blossom 62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 1 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as:$\frac{{Total}\quad{number}\quad{of}\quad{identical}\quad{matches}}{\begin{matrix}\left\lbrack {{length}\quad{of}\quad{the}\quad{longer}\quad{sequence}\quad{plus}\quad{the}\quad{number}\quad{of}\quad{gaps}} \right. \\{{introduced}\quad{into}\quad{the}\quad{longer}\quad{sequence}\quad{in}\quad{order}\quad{to}\quad{align}\quad{the}} \\\left. {{two}\quad{sequences}} \right\rbrack\end{matrix}} \times 100$ TABLE 1 A R N D C Q E G H I L K M F P S T W YV A 4 R −1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 00 2 −4 2 5 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3−1 −3 −3 −4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2−1 −3 −2 5 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3−3 −1 0 0 −3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 10 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1−2 −1 1 5 W − −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 − 1 −4 −3 −2 11 Y −2 −2−2 −3 −2 −1 −2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3−3 3 1 −2 1 −1 −2 −2 0 −3 −1 4

Sequence identity of polynucleotide molecules is determined by similarmethods using a ratio as disclosed above.

Substantially homologous proteins and polypeptides are characterized ashaving one or more amino acid substitutions, deletions or additions.These changes are preferably of a minor nature, that is conservativeamino acid substitutions (see Table 2) and other substitutions that donot significantly affect the folding or activity of the protein orpolypeptide. Also claimedmall deletions of SEQ ID NO:2, typically of oneto about 30 amino acids; and small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue, a small linkerpeptide of up to about 20-25 residues, or a small extension thatfacilitates purification (an affinity tag), such as a poly-histidinetract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al.,Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith andJohnson, Gene 67:31, 1988), or other antigenic epitope or bindingdomain. See, in general Ford et al., Protein Expression and Purification2: 95-107, 1991. DNAs encoding affinity tags are available fromcommercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.). TABLE2 Conservative amino acid substitutions Basic: arginine lysine histidineAcidic: glutamic acid aspartic acid Polar: glutamine asparagineHydrophobic: leucine isoleucine valine Aromatic: phenylalaninetryptophan tyrosine Small: glycine alanine serine threonine methionine

Essential amino acids in the receptor polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al., Proc.Natl. Acad. Sci. USA 88:4498-4502, 1991). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity (e.g., ligand binding and signal transduction) to identifyamino acid residues that are critical to the activity of the molecule.Sites of ligand-receptor interaction can also be determined by analysisof crystal structure as determined by such techniques as nuclearmagnetic resonance, crystallography or photoaffinity labeling. See, forexample, de Vos et al., Science 255:306-312, 1992; Smith et al., J. Mol.Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992.The identities of essential amino acids can also be inferred fromanalysis of homologies with related receptors.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988)

Mutagenesis methods as disclosed above can be combined withhigh-throughput screening methods to detect activity of cloned,mutagenized receptors in host cells. Preferred assays in this regardinclude cell proliferation assays and biosensor-based ligand-bindingassays, which are described below. Mutagenized DNA molecules that encodeactive receptors or portions thereof (e.g., ligand-binding fragments)can be recovered from the host cells and rapidly sequenced using modernequipment. These methods allow the rapid determination of the importanceof individual amino acid residues in a polypeptide of interest, and canbe applied to polypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art canprepare a variety of polypeptides that are substantially homologous toresidues 30 to 250 of SEQ ID NO:2 or allelic variants thereof and retainthe ligand-binding properties of the wild-type receptor. Suchpolypeptides may include additional amino acids from an extracellularligand-binding domain of a Zcytor7 receptor as well as part or all ofthe transmembrane and intracellular domains. Such polypeptides may alsoinclude additional polypeptide segments as generally disclosed above.

Polynucleotides, generally a cDNA sequence, of the present inventionencode the above-described polypeptides. A cDNA sequence which encodes apolypeptide of the present invention is comprised of a series of codons,each amino acid residue of the polypeptide being encoded by a codon andeach codon being comprised of three nucleotides. The amino acid residuesare encoded their respective codons as follows.

-   -   Alanine (Ala) is encoded by GCA, GCC, GCG or GCT;    -   Cysteine (Cys) is encoded by TGC or TGT;    -   Aspartic acid (Asp) is encoded by GAC or GAT;    -   Glutamic acid (Glu) is encoded by GAA or GAG;    -   Phenylalanine (Phe) is encoded by TTC or TTT;    -   Glycine (Gly) is encoded by GGA, GGC, GGG or GGT;    -   Histidine (His) is encoded by CAC or CAT;    -   Isoleucine (Ile) is encoded by ATA, ATC or ATT;    -   Lysine (Lys) is encoded by AAA, or AAG;    -   Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT;    -   Methionine (Met) is encoded by ATG;    -   Asparagine (Asn) is encoded by AAC or AAT;    -   Proline (Pro) is encoded by CCA, CCC, CCG or CCT;    -   Glutamine (Gin) is encoded by CAA or CAG;    -   Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT;    -   Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or TCT;    -   Threonine (Thr) is encoded by ACA, ACC, ACG or ACT;    -   Valine (Val) is encoded by GTA, GTC, GTG or GTT;    -   Tryptophan (Trp) is encoded by TGG; and    -   Tyrosine (Tyr) is encoded by TAC or TAT.

It is to be recognized that according to the present invention, when acDNA is claimed as described above, it is understood that what isclaimed are both the sense strand, the anti-sense strand, and the DNA asdouble-stranded having both the sense and anti-sense strand annealedtogether by their respective hydrogen bonds. Also claimed is themessenger RNA (mRNA) encodes the polypeptides of the present invention,and which mRNA is encoded by the above the above-described cDNA. Amessenger RNA (mRNA) will encode a polypeptide using the same codons asthose defined above, with the exception that each thymine nucleotide (T)is replaced by a uracil nucleotide (U).

The receptor polypeptides of the present invention, includingfull-length receptors, receptor fragments (e.g. ligand-bindingfragments), and fusion polypeptides can be produced in geneticallyengineered host cells according to conventional techniques. Suitablehost cells are those cell types that can be transformed or transfectedwith exogenous DNA and grown in culture, and include bacteria, fungalcells, and cultured higher eukaryotic cells. Eukaryotic cells,particularly cultured cells of multicellular organisms, are preferred.Techniques for manipulating cloned DNA molecules and introducingexogenous DNA into a variety of host cells are disclosed by Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al.,ibid.

The polynucleotides of the present invention can be synthesized usinggene machines. Currently the method of choice is the phosphoramiditemethod. If chemically synthesized double stranded DNA is required for anapplication such as the synthesis of a gene or a gene fragment, theneach complementary strand is made separately. The production of shortgenes (60 to 80 bp) is technically straightforward and can beaccomplished by synthesizing the complementary strands and thenannealing them. For the production of longer genes (>300 bp), however,special strategies must be invoked, because the coupling efficiency ofeach cycle during chemical DNA synthesis is seldom 100%. To overcomethis problem, synthetic genes (double-stranded) are assembled in modularform from single-stranded fragments that are from 20 to 100 nucleotidesin length.

One method for building a synthetic gene requires the initial productionof a set of overlapping, complementary oligonucleotides, each of whichis between 20 to 60 nucleotides long. The sequences of the strands areplanned so that, after annealing, the two end segments of the gene arealigned to give blunt ends. Each internal section of the gene hascomplementary 3′ and 5′ terminal extensions that are designed to basepair precisely with an adjacent section. Thus, after the gene isassembled, the only remaining requirement to complete the process issealing the nicks along the backbones of the two strands with T4 DNAligase. In addition to the protein coding sequence, synthetic genes canbe designed with terminal sequences that facilitate insertion into arestriction endonuclease sites of a cloning vector and other sequencesshould also be added that contain signals for the proper initiation andtermination of transcription and translation.

An alternative way to prepare a full-size gene is to synthesize aspecified set of overlapping oligonucleotides (40 to 100 nucleotides).After the 3′ and 5′ extensions (6 to 10 nucleotides) are annealed, largegaps still remain, but the base-paired regions are both long enough andstable enough to hold the structure together. The duplex is completedand the gaps filled by enzymatic DNA synthesis with E. coli DNApolymerase I. This enzyme uses the 3′-hydroxyl groups as replicationinitiation points and the single-stranded regions as templates. Afterthe enzymatic synthesis is completed, the nicks are sealed with T4 DNAligase. For larger genes (≧1,000 base pairs), the complete gene sequenceis usually assembled from double-stranded fragments that are each puttogether by joining four to six overlapping oligonucleotides (20 to 60bp each). If there is a sufficient amount of the double-strandedfragments after each synthesis and annealing step, they are simplyjoined to one another. Otherwise, each fragment is cloned into a vectorto amplify the amount of DNA available. In both cases, thedouble-stranded constructs are sequentially linked to one another toform the entire gene sequence. Because it is absolutely essential that achemically synthesized gene have the correct sequence of nucleotides,each double-stranded fragment and then the complete sequence ischaracterized by DNA sequence analysis. See Glick, Bernard R. and JackJ. Pasternak, Molecular Biotechnology, Principles & Applications ofRecombinant DNA, (ASM Press, Washington, D.C. 1994), Itakura, K. et al.Synthesis and use of synthetic oligonucleotides. Annu. Rev. Biochem. 53:323-356 (1984), and Climie, S. et al. Chemical synthesis of thethymidylate synthase gene. Proc. Natl. Acad. Sci. USA 87:633-637 (1990).

A DNA sequence encoding a ZCytor7 receptor polypeptide can then beoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are described in theliterature and are available through commercial suppliers.

To direct a Zcytor7 receptor polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of the receptor, or may bederived from another secreted protein (e.g., t-PA) or synthesized denovo. The secretory signal sequence is joined to the ZCytor7 DNAsequence in the correct reading frame. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain signal sequences may be positioned elsewherein the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

Cultured mammalian cells are preferred hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., eds., Current Protocols in MolecularBiology, John Wiley and Sons, Inc., NY, 1987), and liposome-mediatedtransfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al.,Focus 15:80, 1993. The production of recombinant polypeptides incultured mammalian cells is disclosed, for example, by Levinson et al.,U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiteret al., U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134.Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650),COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No.CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol.36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-Ki; ATCC No. CCL 61)cell lines. Additional suitable cell lines are known in the art andavailable from public depositories such as the American Type CultureCollection, Rockville, Md. In general, strong transcription promotersare preferred, such as promoters from SV-40 or cytomegalovirus. See,e.g., U.S. Pat. No. 4,956,288. Other suitable promoters include thosefrom metallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) andthe adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems mayalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used.

Other higher eukaryotic cells can also be used as hosts, includinginsect cells, plant cells and avian cells. Transformation of insectcells and production of foreign polypeptides therein is disclosed byGuarino et al., U.S. Pat. No. 5,162,222; Bang et al., U.S. Pat. No.4,775,624; and WIPO publication WO 94/06463. The use of Agrobacteriumrhizogenes as a vector for expressing genes in plant cells has beenreviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.

Fungal cells, including yeast cells, and particularly cells of the genusSaccharomyces, can also be used within the present invention, such asfor producing receptor fragments or polypeptide fusions. Methods fortransforming yeast cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake,U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; andMurray et al., U.S. Pat. No. 4,845,075. Transformed cells are selectedby phenotype determined by the selectable marker, commonly drugresistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A preferred vector system for use in yeast isthe POT1 vector system disclosed by Kawasaki et al. (U.S. Pat. No.4,931,373), which allows transformed cells to be selected by growth inglucose-containing media. Suitable promoters and terminators for use inyeast include those from glycolytic enzyme genes (see, e.g., Kawasaki,U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; andBitter, U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. Seealso U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromycesfragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986 andCregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349.Methods for transforming Acremonium chrysogenum are disclosed by Suminoet al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora aredisclosed by Lambowitz, U.S. Pat. No. 4,486,533.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell.

Within one aspect of the present invention, a novel receptor is producedby a cultured cell, and the cell is used to screen for ligands for thereceptor, including the natural ligand, as well as agonists andantagonists of the natural ligand. To summarize this approach, a cDNA orgene encoding the receptor is combined with other genetic elementsrequired for its expression (e.g., a transcription promoter), and theresulting expression vector is inserted into a host cell. Cells thatexpress the DNA and produce functional receptor are selected and usedwithin a variety of screening systems.

Mammalian cells suitable for use in expressing ZCytor7 receptors andtransducing a receptor-mediated signal include cells that express otherreceptor subunits which may form a functional complex with Zcytor7.These subunits may include those of the interferon receptor family or ofother class II or class I cytokine receptors. It is also preferred touse a cell from the same species as the receptor to be expressed. Withina preferred embodiment, the cell is dependent upon an exogenouslysupplied hematopoietic growth factor for its proliferation. Preferredcell lines of this type are the human TF-1 cell line (ATCC numberCRL-2003) and the AML-193 cell line (ATCC number CRL-9589), which areGM-CSF-dependent human leukemic cell lines. In the alternative, suitablehost cells can be engineered to produce the necessary receptor subunitor other cellular component needed for the desired cellular response.For example, the murine cell line BaF3 (Palacios and Steinmetz, Cell 41:727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135,1986) or a baby hamster kidney (BHK) cell line can be transfected toexpress the necessary □ subunit (also known as KH97) as well as aZCytor7 receptor. The latter approach is advantageous because cell linescan be engineered to express receptor subunits from any species, therebyovercoming potential limitations arising from species specificity. Inthe alternative, species orthologs of the human receptor cDNA can becloned and used within cell lines from the same species, such as a mousecDNA in the BaF3 cell line. Cell lines that are dependent upon onehematopoietic growth factor, such as GM-CSF, can thus be engineered tobecome dependent upon a ZCytor7 ligand.

Cells expressing functional receptor are used within screening assays. Avariety of suitable assays are known in the art. These assays are basedon the detection of a biological response in a target cell. One suchassay is a cell proliferation assay. Cells are cultured in the presenceor absence of a test compound, and cell proliferation is detected by,for example, measuring incorporation of tritiated thymidine or bycalorimetric assay based on the metabolic breakdown of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MT)(Mosman, J. Immunol. Meth. 65: 55-63, 1983). An alternative assay formatuses cells that are further engineered to express a reporter gene. Thereporter gene is linked to a promoter element that is responsive to thereceptor-linked pathway, and the assay detects activation oftranscription of the reporter gene. A preferred promoter element in thisregard is a serum response element, or SRE (see, e.g., Shaw et al., Cell56:563-572, 1989). A preferred such reporter gene is a luciferase gene(de Wet et al., Mol. Cell. Biol. 7:725, 1987). Expression of theluciferase gene is detected by luminescence using methods known in theart (e.g., Baumgartner et al., J. Biol. Chem. 269:29094-29101, 1994;Schenborn and Goiffin, Promega Notes 41:11, 1993). Luciferase activityassay kits are commercially available from, for example, Promega Corp.,Madison, Wis. Target cell lines of this type can be used to screenlibraries of chemicals, cell-conditioned culture media, fungal broths,soil samples, water samples, and the like. For example, a bank ofcell-conditioned media samples can be assayed on a target cell toidentify cells that produce ligand. Positive cells are then used toproduce a cDNA library in a mammalian expression vector, which isdivided into pools, transfected into host cells, and expressed. Mediasamples from the transfected cells are then assayed, with subsequentdivision of pools, re-transfection, subculturing, and re-assay ofpositive cells to isolate a cloned cDNA encoding the ligand.

A natural ligand for the ZCytor7 receptor can also be identified bymutagenizing a cell line expressing the receptor and culturing it underconditions that select for autocrine growth. See WIPO publication WO95/21930. Within a typical procedure, BaF3 cells expressing ZCytor7 andthe necessary additional subunits are mutagenized, such as with2-ethylmethanesulfonate (EMS). The cells are then allowed to recover inthe presence of IL-3, then transferred to a culture medium lacking IL-3and IL-4. Surviving cells are screened for the production of a ZCytor7ligand, such as by adding soluble receptor to the culture medium or byassaying conditioned media on wild-type BaF3 cells and BaF3 cellsexpressing the receptor.

An additional screening approach provided by the present inventionincludes the use of hybrid receptor polypeptides. These hybridpolypeptides fall into two general classes. Within the first class, theintracellular domain of Z-Cytor7, comprising approximately residues 275to 553 of SEQ ID NO:2, is joined to the ligand-binding domain of asecond receptor. It is preferred that the second receptor be ahematopoietic cytokine receptor, such as mpl receptor (Souyri et al.,Cell 63: 1137-1147, 1990). The hybrid receptor will further comprise atransmembrane domain, which may be derived from either receptor. A DNAconstruct encoding the hybrid receptor is then inserted into a hostcell. Cells expressing the hybrid receptor are cultured in the presenceof a ligand for the binding domain and assayed for a response. Thissystem provides a means for analyzing signal transduction mediated byZCytor7 while using readily available ligands. This system can also beused to determine if particular cell lines are capable of responding tosignals transduced by ZCytor7. A second class of hybrid receptorpolypeptides comprise the extracellular (ligand-binding) domain ofZCytor7 (approximately residues 30 to 250 of SEQ ID NO:2) with anintracellular domain of a second receptor, preferably a hematopoieticcytokine receptor, and a transmembrane domain. Hybrid receptors of thissecond class are expressed in cells known to be capable of responding tosignals transduced by the second receptor. Together, these two classesof hybrid receptors enable the identification of a responsive cell typefor the development of an assay for detecting a Zcytor7 ligand.

Cells found to express the ligand are then used to prepare a cDNAlibrary from which the ligand-encoding cDNA can be isolated as disclosedabove. The present invention thus provides, in addition to novelreceptor polypeptides, methods for cloning polypeptide ligands for thereceptors.

The tissue specificity of Zcytor7 expression suggests a role in thedevelopment of the kidney, pancreas, prostate or nervous tissues. Inview of the tissue specificity observed for this receptor, agonists(including the natural ligand) and antagonists have enormous potentialin both in vitro and in vivo applications. Compounds identified asreceptor agonists are useful for stimulating proliferation anddevelopment of target cells in vitro and in vivo. For example, agonistcompounds are useful as components of defined cell culture media, andmay be used alone or in combination with other cytokines and hormones toreplace serum that is commonly used in cell culture. Agonists may beuseful in specifically promoting the growth and/or development ofnervous, pancreatic or prostate-derived cells in culture. Antagonistsare useful as research reagents for characterizing sites ofligand-receptor interaction. In vivo, receptor agonists or antagonistsmay find application in the treatment of renal, neural, pancreatic orprostate diseases.

ZCytor7 may also be used within diagnostic systems for the detection ofcirculating levels of ligand. Within a related embodiment, antibodies orother agents that specifically bind to ZCytor7 can be used to detectcirculating receptor polypeptides. Elevated or depressed levels ofligand or receptor polypeptides may be indicative of pathologicalconditions, including cancer.

ZCytor7 receptor polypeptides can be prepared by expressing a truncatedDNA encoding the extracellular domain, for example, a polypeptide whichcontains residues 30 through 250 of a human ZCytor7 receptor (SEQ IDNO:2) or the corresponding region of a non-human receptor. It ispreferred that the extracellular domain polypeptides be prepared in aform substantially free of transmembrane and intracellular polypeptidesegments. For example, the C-terminus of the receptor polypeptide may beat residue 250 of SEQ ID NO:2 or the corresponding region of an allelicvariant or a non-human receptor. To direct the export of the receptordomain from the host cell, the receptor DNA is linked to a second DNAsegment encoding a secretory peptide, such as a t-PA secretory peptide.To facilitate purification of the secreted receptor domain, a C-terminalextension, such as a poly-histidine tag, substance P, Flag™ peptide(Hopp et al., Biotechnology 6:1204-1210, 1988; available from EastmanKodak Co., New Haven, Conn.) or another polypeptide or protein for whichan antibody or other specific binding agent is available, can be fusedto the receptor polypeptide.

In an alternative approach, a receptor extracellular domain can beexpressed as a fusion with immunoglobulin heavy chain constant regions,typically an F_(C) fragment, which contains two constant region domainsand a hinge region but lacks the variable region. Such fusions aretypically secreted as multimeric molecules wherein the F_(C) portionsare disulfide bonded to each other and two receptor polypeptides arearrayed in closed proximity to each other. Fusions of this type can beused to affinity purify the cognate ligand from solution, as an in vitroassay tool, to block signals in vitro by specifically titrating outligand, and as antagonists in vivo by administering them parenterally tobind circulating ligand and clear it from the circulation. To purifyligand, a ZCytor7-Ig chimera is added to a sample containing the ligand(e.g., cell-conditioned culture media or tissue extracts) underconditions that facilitate receptor-ligand binding (typicallynear-physiological temperature, pH, and ionic strength). Thechimera-ligand complex is then separated by the mixture using protein A,which is immobilized on a solid support (e.g., insoluble resin beads).The ligand is then eluted using conventional chemical techniques, suchas with a salt or pH gradient. In the alternative, the chimera itselfcan be bound to a solid support, with binding and elution carried out asabove. Chimeras with high binding affinity are administered parenterally(e.g., by intramuscular, subcutaneous or intravenous injection).Circulating molecules bind ligand and are cleared from circulation bynormal physiological processes. For use in assays, the chimeras arebound to a support via the F_(C) region and used in an ELISA format.

A preferred assay system employing a ligand-binding receptor fragmentuses a commercially available biosensor instrument (BIAcore™, PharmaciaBiosensor, Piscataway, N.J.), wherein the receptor fragment isimmobilized onto the surface of a receptor chip. Use of this instrumentis disclosed by Karlsson, J. Immunol. Methods 145:229-240, 1991 andCunningham and Wells, J. Mol. Biol. 234:554-563, 1993. A receptorfragment is covalently attached, using amine or sulfhydryl chemistry, todextran fibers that are attached to gold film within the flow cell. Atest sample is passed through the cell. If ligand is present in thesample, it will bind to the immobilized receptor polypeptide, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination of on- and off-rates, from which binding affinity canbe calculated, and assessment of stoichiometry of binding.

Ligand-binding receptor polypeptides can also be used within other assaysystems known in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see, Scatchard, Ann. NY Acad. Sci.51: 660-672, 1949) and calorimetric assays (Cunningham et al., Science253:545-548, 1991; Cunningham et al., Science 254:821-825, 1991).

A receptor ligand-binding polypeptide can also be used for purificationof ligand. The receptor polypeptide is immobilized on a solid support,such as beads of agarose, cross-linked agarose, glass, cellulosicresins, silica-based resins, polystyrene, cross-linked polyacrylamide,or like materials that are stable under the conditions of use. Methodsfor linking polypeptides to solid supports are known in the art, andinclude amine chemistry, cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, and hydrazide activation. The resulting media will generallybe configured in the form of a column, and fluids containing ligand arepassed through the column one or more times to allow ligand to bind tothe receptor polypeptide. The ligand is then eluted using changes insalt concentration or pH to disrupt ligand-receptor binding.

ZCytor7 polypeptides can also be used to prepare antibodies thatspecifically bind to ZCytor7 polypeptides. As used herein, the term“antibodies” includes polyclonal antibodies, monoclonal antibodies,single-chain antibodies and antigen-binding fragments thereof such asF(ab′)₂ and Fab fragments, and the like, including geneticallyengineered antibodies. Antibodies are defined to be specifically bindingif they bind to a ZCytor7 polypeptide with a K_(a) of greater than orequal to 10⁷/M. The affinity of a monoclonal antibody can be readilydetermined by one of ordinary skill in the art (see, for example,Scatchard, ibid.).

Methods for preparing polyclonal and monoclonal antibodies are wellknown in the art (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; andHurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press, Inc., Boca Raton, Fla., 1982). As would beevident to one of ordinary skill in the art, polyclonal antibodies canbe generated from a variety of warm-blooded animals such as horses,cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. Theimmunogenicity of a ZCytor7 polypeptide may be increased through the useof an adjuvant such as Freund's complete or incomplete adjuvant. Avariety of assays known to those skilled in the art can be utilized todetect antibodies which specifically bind to ZCytor7 polypeptides.Exemplary assays are described in detail in Antibodies: A LaboratoryManual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press,1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations,enzyme-linked immunosorbent assays (ELISA), dot blot assays, inhibitionor competition assays, and sandwich assays.

Antibodies to ZCytor7 may be used for tagging cells that express thereceptor, for affinity purification, within diagnostic assays fordetermining circulating levels of soluble receptor polypeptides, and asantagonists to block ligand binding and signal transduction in vitro andin vivo.

Anti-idiotypic antibodies which bind to the antigenic binding site ofantibodies to Zcytor7 are also considered part of the present invention.The antigenic binding region of the anti-idiotypic antibody thus willmimic the ligand binding region of Zcytor7. An anti-idiotypic antibodythus could be used to screen for possible ligands of the Zcytor7receptor. Thus neutralizing antibodies to Zcytor7 can be used to produceanti-idiotypic antibodies by methods well known in the art as isdescribed in, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, (Cold Spring Harbor, N.Y., 1989); andHurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques andApplications, (CRC Press, Inc., Boca Raton, Fla., 1982).

Zcytor-7 maps 795.76 cR from the top of the human chromosome 6 linkagegroup on the WICGR radiation hybrid map. Relative to the centromere, itsnearest proximal marker was CHLC.GATA32B03 and its nearest distal makerwas SGC32063. The use of surrounding markers also helped positionzcytor-7 in the 6q22-q23 region on the CHLC chromosome 6 version v8c7integrated marker map. The locus where Zcytor7 maps onto chromosome 6 isa common breakpoint area in ALL (acute lymphoblastic leukemia) and NHL(non-Hodgkin lymphoma) as well as in AML (acute myelogenous leukemia)and CML (chronic myeloid leukemia). It is interesting to note that theMYB (avian myeloblastosis viral oncogene homolog) gene, which encodesproteins critical for hematopoetic. cell proliferation and development,appears to be less than 800 kB from zcytor7. The 6q-deletion breakpointsoccur slightly distal to the MYB gene and although the neoplasms showhigh levels of MYB mRNA, the gene itself appears to be intact.

Thus Zcytor7 could be used to generate a probe that could allowdetection of an aberration of the Zctyor7 gene in the 6q chromosomewhich may indicate the presence of a cancerous cell such as leukemiccells which may still be present in after chemical or radiation therapy.If the Zcytor7 gene is deleted by the chromosomal abnormality, only onecopy can be used to determine whether one or two copies of the gene arepresent per nucleus, thus indicating the percentage cancerous cellsmight be present relative to normal cells. For further discussions ondeveloping polynucleotide probes and hybridization see Current Protocolsin Molecular Biology Ausubel, F. et al. Eds. (John Wiley & Sons Inc.1991).

Pharmaceutical Compositions

Pharmaceutical compositions can be formulated which contain the solublereceptor, antibody or anti-idiotypic antibodies of the presentinvention. Generally included in such protein therapeutic compositionsare buffers; surface adsorption inhibitors such as surfactants andpolyols; and isotonic amounts of a physiologically acceptable salt. Thecomposition may be formulated as an aqueous solution or a lyophilizedpowder. The latter is reconstituted prior to use with a pharmaceuticallyacceptable diluent such as sterile water for injection.

Examples of buffers which can be used for the above-describedpharmaceutical compositions include low ionic strength, physiologicallyacceptable buffers that are effective within the pH range of 5.0-7.0.Such buffers include phosphate, acetate, citrate, succinate andhistidine buffers.

Examples of surface adsorption inhibitors which can be used in theabove-described pharmaceutical compositions include non-ionicsurfactants and polyols. Non-ionic surfactants include polyoxyethylenesorbitan fatty acid esters, such as polysorbate 20 (polyoxyethylenesorbitan monolaurate), and the like. Other non-ionic surfactants usefulin this regard include polyethylene oxides; sorbitan esters;polyoxyethylene alkyl ethers; and glycerides of fatty acid, includingglyceryl monooleate and glyceryl monostearate. Polyols which can be usedinclude polyethylene glycol, e.g. PEG 3350, mannitol, xylitol, sorbitol,inositol, and glycerol. In general, the surface adsorption inhibitorwill be included within the composition at a concentration from 0.001%to 5%.

Physiologically acceptable salts are generally included in a proteintherapeutic composition generally in an amount isotonic to human blood.Preferred salts in this regard include chloride salts such as NaCl, KCl,CaCl₂ and MgCl₂.

Albumin may also be included in the above-described pharmaceuticalcompositions. Human serum albumin is preferred for inclusion inpharmaceutical compositions intended for human use. Albumin is useful asan excipient in lyophilized compositions and acts as a stabilizer whenincluded at a concentration of 0.1-1.0%. Albumin may useful as a surfaceadsorption inhibitor.

One or more preservatives may also be included in the pharmaceuticalcompositions of the present invention. Common preservative includemethylparaben, propylparaben, benzyl alcohol, m-cresol,ethylmercurithiosalycilate, phenol, thimerosol and the like. Methods offormulation of pharmaceutical compositions are well known in the art andare disclosed, for example, in Remington's Pharmaceutical Sciences,Gennaro, ed., (Mack Publishing Co., Easton, Pa., 1996) Dosages

Therapeutic doses of the protein compositions of the present inventionwill generally be in the range of 0.1 to 100 μg/kg of patient per daywith the exact dose determined by the clinician.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLE 1 Cloning of Zcytor7

Expressed sequence tag (EST) 277139 (SEQ ID NO:3) was identified. ThecDNA clone (ID No. 50416) was obtained from the IMAGE consortiumLawrence Livermore National Laboratory through Genome Systems, Inc. ThecDNA was supplied as an agar stab containing E. coli transfected with aplasmid having the cDNA of interest. The E. coli was streaked on an agarplate. The plasmid was designated pSL7139. The cDNA insert in plasmidpSL7139 was sequenced. The insert was determined to be 1231 bp inlength, but was not a full length sequence.

A human testis cDNA template was made using a MARATHON™ cDNAAmplification Kit (Clontech Laboratories, Inc., Palo Alto, Calif.)according to the supplier

s instructions. A 5

RACE reaction was used to obtain a full-length cDNA. The RACE reactionwas carried out in two reactions employing two sets of primers. ReactionI (outer nest), using primers ZC11,107 (SEQ ID NO:4) and AP-1 (SEQ IDNO: 5) (Clontech Laboratories) was run for 35 cycles at 98° C. for 20seconds, 45° C. for 20 seconds; 68° for 4 minutes and a final extensiontime of 10 minutes at 68° C. One μl of a 1:100 dilution of the reactionproduct was used as a template in reaction II (inner nest). Primers wereZC11,108 (SEQ ID NO:6) and AP-2 (SEQ ID NO:7) (Clontech Laboratories).The reaction was run at 98° C. for 30 seconds, and 30 cycles each cyclebeing comprised of 98° C. for 28 seconds; 43° C. for 20 seconds; and 68°C. for 3.5 minutes with a final extension at 68° C. for 10 minutes.

The product of the inner nest RACE reaction was subcloned using aPCR-SCRIPT™ kit (Stratagene Cloning Systems, La Jolla, Calif.) toprepare the plasmid pSLR7-1. Sequence analysis of this plasmid indicatedthat the 5

RACE-generated sequence extended the sequence of pSL7139 by 555 bp.

Full-length cDNA was obtained by screening a λZAP® human testis cDNAlibrary using a probe that was generated by PCR primers ZC11,526 (SEQ IDNO:9) and ZC11,108 (SEQ ID NO:6) and pSLR7-1 as template and thenre-amplified. The resulting probe was purified through recovery fromlow-melt agarose gel electrophoresis and was labeled with 32P-α-dCTPusing a MEGAPRIME™ labeling kit (Amersham Corp., Arlington, Heights,Ill.). The labeled probe was purified on a push column (NUCTRAP® probepurification column; Stratagene Cloning Systems).

The first strand cDNA reaction contained 15 μl of human testis twicepoly d(T)-selected poly (A)⁺ mRNA (Clontech Laboratories) at aconcentration of 1.0 μg/μl, and 3 μl of 20 pmole/μl first strand primerZC6091 (SEQ ID NO:8) containing an Xho I restriction site. The mixturewas heated at 70° C. for 4 minutes and cooled by chilling on ice. Firststand cDNA synthesis was initiated by the addition of 12 μl of firststrand buffer (5×SUPERSCRIP™ buffer; Life Technologies, Gaithersburgh,Md.), 6 μl of 100 mM dithiothreitol, 3 μl of deoxynucleotidetriphosphate solution containing 10 mM each of dTTP, dATP, dGTP, and5-methyl-dCTP (Pharmacia LKB Biotechnology, Piscataway, N.J.) to theRNA-primer mixture. The reaction mixture was incubated at 37° C. for 2minutes, followed by the addition of 15 μl of 200 U/μl Rnase H⁻ reversetranscriptase (SUPERSCRIPT II^(<<); Life Technologies). The efficiencyof the first strand synthesis was analyzed in a parallel reaction by theaddition of 5 μCi of ³²P-αdCTP to 5 μl aliquot from one of the reactionmixtures to label the reaction for analysis. The reactions wereincubated at 37° C. for 10 minutes, 45° C. for 1 hour, then incubated at50° C. for 10 minutes. Unincorporated ³²P-αdCTP in the labeled reactionand the unincorporated nucleotides and primers in the unlabeled firststrand reactions were removed by chromatography on a 400 pore size gelfiltration column (Clontech Laboratories). The length of labeled firststrand cDNA was determined by agarose gel electrophoresis.

The second strand reaction contained 120 μl of the unlabeled firststrand cDNA, 36 μl of 5×polymerase I buffer (125 mM Tris: HCl, pH 7.5,500 mM KCl, 25 mM MgCl₂, 50 mM (NH₄)₂SO₄)), 2.4 μl of 100 mMdithiothreitol, 3.6 μl of a solution containing 10 mM of eachdeoxynucleotide triphosphate, 6 μl of 5 mM β-NAD, 3.6 μL of 3 U/μl E.coli DNA ligase (New England Biolabs), 9 μl of 10 U/μl E. coli DNApolymerase I (New England Biolabs), and 1.8 μl of 2 U/μl RNase H (lifeTechnologies). A 10 μl aliquot from one of the second strand synthesisreactions was labeled by the addition of 10 μCi ³²P-αdCTP to monitor theefficiency of second strand synthesis. The reactions were incubated at16° C. for two hours, followed by the addition of 15 μl T4 DNApolymerase (10 U/μl, Boerhinger Mannheim, Indianapolis, Ind.) andincubated for an additional 5 minutes at 16° C. Unincorporated ³²P-αdCTPin the labeled reaction was removed by chromatography through a 400 poresize gel filtration (Clontech Laboratories) before analysis by agarosegel electrophoresis. The unlabeled second strand reaction was terminatedby the addition of 20 μl 0.5 M EDTA and extraction withphenol/chloroform and chloroform followed by ethanol precipitation inthe presence of 2.5 M ammonium acetate and 4 μg of glycogen carrier. Theyield of cDNA was estimated to be approximately 3 μg from starting mRNAtemplate of 15 μg.

Eco RI adapters were ligated onto the 5

ends of the cDNA described above to enable cloning into an expressionvector. A 10 μl aliquot of cDNA (approximately 1.5 μg) and 5 μof 65pmole/μl of Eco RI adapter (Pharmacia LKB Biotechnology Inc.) were mixedwith 2 μl 10× ligase buffer (660 mM Tris-HCl pH 7.5, 100 mM MgCl₂), 2 μlof 10 mM ATP and 1 μl of 15.U/μl T4 DNA ligase (Promega Corp., Madison,Wis.). The reaction was incubated 2 hours at 5° C., two hours at 7.5°C., 2 hours at 10° C, and 10 hours at 12.5° C. The reaction wasterminated by incubation at 70° C. for 20 minutes.

To facilitate the directional cloning of the cDNA into an expressionvector, the cDNA was digested with Xho I, resulting in a cDNA having a 5

Eco RI cohesive end and a 3

Xho cohesive end. The Xho I restriction site at the 3

end of the cDNA had been previously introduced using the ZC6091 primer(SEQ ID NO: 8). Restriction enzyme digestion was carried out in areaction mixture containing 20 μl of cDNA as described above, 10 μl of10×H Buffer Xho I (Boehringer Mannheim), 69 μl H₂O, and 1.0 μl of 40U/μl Xho I (Boehringer Mannheim). Digestion was carried out at 37° C.for 40 minutes. The reaction was terminated by incubation at 70° C. for10 minutes and chromatography through a 400 pore size gel filtrationcolumn (Clontech Laboratories).

The cDNA was ethanol precipitated, washed with 70% ethanol, air driedand resuspended in 14 μl water, 2 μl of ligase buffer (Promega Corp.,Madison, Wis.), 2 μl T4 polynucleotide kinase (10 U/μl, LifeTechnologies). Following incubation at 37° C. for 30 minutes, the cDNAwas heated to 65° C. for 5 minutes, cooled on ice, and electrophoresedon a 0.8% low melt agarose gel. The contaminating adapters and cDNAbelow 0.6 kb in length were excised from the gel. The electrodes werereversed, and the cDNA was electrophoresed until concentrated near thelane origin. The area of the gel containing the concentrated cDNA wasexcised and placed in a microfuge tube, and the approximate volume ofthe gel slice was determined. An aliquot of water approximately threetimes the volume of the gel slice (300 μl) and 35 μl 10×β-agarose Ibuffer (New England Biolabs) were added to the tube, and the agarose wasmelted by heating to 65° C. for 15 minutes. Following equilibration ofthe sample to 45° C., 3 μl of 1 U/μl β-agarose I (New England Biolabs)was added, and the mixture was incubated for 60 minutes at 45° C. todigest the agarose. After incubation, 40 μl of 3 M Na acetate was addedto the sample, and the mixture was incubated on ice for 15 minutes. Thesample was centrifuged at 14,000×g for 15 minutes at room temperature toremove undigested agarose. The cDNA was ethanol precipitated, washed in70% ethanol, air-dried and resuspended in 10 μl water.

The resulting cDNA was cloned into the lambda phage vector λZap^(<<) II(Stratagene Cloning Systems) that was predigested with Eco RI and Xho Iand dephosphorylated. Ligation of the cDNA to the λZap<< II vector wascarried out in a reaction mixture containing 1.0 μl of prepared vector,1.0 μl of human testis cDNA, 1.0 μl 10× Ligase Buffer (Promega Corp.),1.0 μl of 10 mM ATP, 5 μl water, and 1.0 μl of T4 DNA Ligase at 15units/ml (Promega Corp.). The ligation mixture was incubated at 5°-15°C. overnight in a temperature gradient. After incubation, the ligationmixture was packaged into phage using an in vitro packaging extract(Gigapack<< III Gold packaging extract; Stratagene Cloning Systems), andthe resulting library was titered according to the manufacturer

s specifications.

The human testis λZAP<< II library was used to infect E. coli host cells(XL1-Blue MRF

strain (Stratagene Cloning Systems), and 1.5×10⁶ plaque forming units(pfu) were plated onto 150-mm NZY plates at a density of about 50,000pfu/plate. The inoculated plates were incubated overnight at 37° C.Filter plaque lifts were made using nylon membranes (Hybond™-N; AmershamCorp., Arlington Heights, Ill.), according to the procedures provided bythe manufacturer. The filters were processed by denaturation in solutioncontaining 1.5 M NaCl and 0.5 M NaOH for 6 minutes at room temperature.The filters were blotted briefly on filter paper to remove excessdenaturation solution, followed by neutralization for 6 minutes in 1 MTris-HCl, pH 7.5, and 1.5 M NaCl. Phage DNA was fixed onto the filterswith 1,200 μJoules of UV energy in a UV Crosslinker (Stratalinker<<;Stratagene Cloning Systems). After fixing, the filters were firstpre-washed in an aqueous solution containing 0.25× standard sodiumcitrate (SSC), 0.25% sodium dodecyl sulfate (SDS) and 1 mM EDTA toremove cellular debris and then prehybridized in hybridization solution(5×SSC, 5× Denhardt

s solution, 0.2% SDS and 1 mM EDTA). Heat-denatured, sheared salmonsperm DNA at a final concentration of 100 μg/ml was added. The filterswere prehybridized at 65° C. overnight.

A probe was prepared as a PCR product by using oligonucleotide primersdesigned to amplify the human Zcytor7 coding region. A PCR reactionmixture was prepared containing 2 μl of ZC11526 (SEQ ID NO:9) 2 μl ofZC11,108 (SEQ ID NO:6), 1 μl of an overnight bacterial culture ofpSLR7-1, 1 μl of 10 mM dNTP, 10 μl of 10× KlenTaq buffer (ClontechLaboratories), 82 μl water, and 2 μl KlenTaq DNA polymerase (Clontechlaboratories). The PCR reaction was run as follows: 94° C. for 1 minute;30 cycles of 95° C. for 20 seconds, 43° C. for 20 seconds, 68° C. for 1minute; then held at 68° C. for 10 minutes. The PCR product wasre-amplified and gel purified on a 0.8% low melt agarose gel.

Fifty nanograms PCR product was radiolabeled with 32P-α-dCTP by randompriming using the MEGAPRIME<< DNA Labeling System (Amersham), accordingto the manufacturer

s specifications. The prehybridization solution was replaced with freshhybridization solution containing 1.4×10⁶ cpm/ml labeled probe andallowed to hybridize for 64 hours at 60° C. After hybridization, thehybridization solution was removed and the filters were rinsed in a washsolution containing 0.25×SSC, 0.25% SDS and 1 mM EDTA at 65° C. Thefilters were placed on autoradiograph film and exposed at −70° C. withintensifying screens for 72 hours.

Examination of the autoradiographs revealed multiple regions thathybridized with labeled probe. Agar plugs were picked from 12 regionsfor purification. Each agar plug was soaked 2 hours in 0.5 ml of SMsolution containing 25 ml 4M NaCl, 10 ml 1M MgSO₄, 25 ml 2M Tris HCl, 5ml 2% gelatin and 935 ml H₂O and 10% (v/v) chloroform (Sambrook et al.Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989). After incubation, thephages from each plug were diluted 1:1000 in SM. Aliquots of 50 μl wereplated on 100 mm plates containing 300 μl of E. coli XL-1 Blue MRF

cells. The plates were incubated overnight at 37° C., and filter liftswere prepared, prehybridized overnight, hybridized overnight with ahybridization solution containing 1.1×10⁶ cpm/ml labeled probe, washedand autoradiographed. Examination of the resulting autoradiographsrevealed 10 positive signals. The positive plaques were subjected to anadditional round of purification.

The plasmids were excised using an ExASSIST/SOLR<< system (StratageneCloning Systems), according to the manufacturer

s specifications. These plasmid inserts were amplified by PCR for sizedetermination. A clone, designated pSLR7-2 was sequenced and determinedto have an insert of 3,532 bp in size.

EXAMPLE 2 Northern Blot Analysis

A 970 bp fragment of the Zcytor7 cDNA containing nucleotides 822-1791was random primer labeled using a MULTIPRIME™ kit (Amersham Corp.).Labeled cDNA was purified from free counts using a push column(Stratagene Cloning Systems). A human RNA master dot blot (ClontechLaboratories) for three hours at 65° C., then hybridized with 10⁶ cpm/mlof labeled cDNA probe. The expression pattern for this blot, whichcontained RNA samples which had been normalized to the mRNA expressionlevels of eight different housekeeping genes, was highest in kidney,followed by spinal cord, prostate, and cerebellum.

EXAMPLE 3 Expression of Human Zcytor7 mRNA in Human Tissues

Poly(A)⁺ RNAs isolated from adrenal cortex, adrenal medulla, brain,colon, heart, kidney, liver, lung, ovary, pancreas, prostate, placenta,peripheral blood leukocytes, stomach, spleen, skeletal muscle, smallintestine, testis, thymus, thyroid, fetal brain, fetal lung, fetal liverand fetal kidney were hybridized under high stringency conditions with aradiolabeled DNA probe containing nucleotides 822-1791 of (SEQ ID NO:1). Membranes were purchased from Clontech. The membrane were washedwith 0.1×SSC, 0.1% SDS at 50° C. and autoradiographed for 24 hours. ThemRNA levels were highest in adrenal cortex, pancreas and prostate withlower levels in testis, stomach, adrenal medulla and thyroid.

EXAMPLE 4 Chromosomal Assignment and Placement of Zcytor-7

Zcytor-7 was mapped to chromosome 6 using the commercially availableversion of the Whitehead Institute/MIT Center for Genome Research's“GeneBridge 4 Radiation Hybrid Panel” (Research Genetics, Inc.,Huntsville, Ala.). The GeneBridge 4 Radiation Hybrid Panel containsPCRable DNAs from each of 93 radiation hybrid clones, plus two controlDNAs (the BFL donor and the A23 recipient). A publicly available WWWserver (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) allowsmapping relative to the Whitehead Institute/MIT Center for GenomeResearch's radiation hybrid map of the human genome (the “WICGR”radiation hybrid map) which was constructed with the GeneBridge 4Radiation Hybrid Panel.

For the mapping of zcytor-7 with the, “GeneBridge 4 RH Panel”, 25 μlreactions were set up in a PCRable 96-well microtiter plate (Stratagene,La Jolla, Calif.) and used in a “RoboCycler Gradient 96” thermal cycler(Stratagene). Each of the 95 PCR reactions consisted of 2.5 μl 50×“Advantage KlenTaq Polymerase Mix” (CLONTECH Laboratories, Inc., PaloAlto, Calif.), 2=|1 dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City,Calif.), 1.25 μl sense primer, ZC 11,131, (SEQ ID NO: 11), 1.25 μlantisense primer, ZC 11,155, (SEQ ID NO: 12), 2.5 μl “RediLoad”(Research Genetics, Inc., Huntsville, Ala.), 0.5=|1 “Advantage KlenTaqPolymerase Mix” (Clontech Laboratories, Inc.), 25 ng of DNA from anindividual hybrid clone or control and water is added to bring up thetotal volume to 25=|1. The reactions were overlaid with an equal amountof mineral oil and sealed. The PCR cycler conditions were as follows: aninitial 1 cycle 4 minute denaturation at 94° C., 35 cycles of a 1 minutedenaturation at 94° C., 1.5 minute annealing at 63° C. and 1.5 minuteextension at 72° C., followed by a final 1 cycle extension of 7 minutesat 72° C. The reactions were separated by electrophoresis on a 3%NuSieve GTG agarose gel (FMC Bioproducts, Rockland, Me.).

The results showed that zcytor-7 maps 795.76 cR from the top of thehuman chromosome 6 linkage group on the WICGR radiation hybrid map.Relative to the centromere, its nearest proximal marker wasCHLC.GATA32BO3 and its nearest distal maker was SGC32063. The use ofsurrounding markers also helped position zcytor-7 in the 6q22-q23 regionon the CHLC chromosome 6 version v8c7 integrated marker map (TheCooperative Human Linkage Center, WWW server:http://www.chlc.org/ChlclntegratedMaps.html) and to 6q22.33-q23.1 on theintegrated LDB chromosome 6 map (The Genetic Location Database,University of Southhampton, WWWserver:http://cedar.genetics.soton.ac.uk/public_html/).

This is a common breakpoint area in ALL (acute lymphoblastic leukemia)and NHL (non-Hodgkin lymphoma) as well as in AML (acute myelogenousleukemia) and CML (chronic myeloid leukemia). It is interesting to notethat the MYB (avian myeloblastosis viral oncogene homolog) gene, whichencodes proteins critical for hematopoetic cell proliferation anddevelopment, appears to be less than 800 kB from zcytor7. The6q-deletion breakpoints occur slightly distal to the MYB gene andalthough the neoplasms show high levels of MYB mRNA, the gene itselfappears to be intact.

1. An isolated polynucleotide encoding a ligand-binding receptorpolypeptide, said polypeptide comprising a sequence of amino acidsselected from the group consisting of: (a) residues 30 to 250 of SEQ IDNO:2; (b) allelic variants of (a); and (c) sequences that are at least80% identical to (a) or (b).
 2. An isolated polynucleotide according toclaim 1 wherein said polypeptide further comprises a transmembranedomain.
 3. An isolated polynucleotide according to claim 2 wherein saidtransmembrane domain comprises residues 251 to 274 of SEQ ID NO:2, or anallelic variant thereof.
 4. An isolated polynucleotide according toclaim 2 wherein said polypeptide further comprises an intracellulardomain.
 5. An isolated polynucleotide according to claim 4 wherein saidintracellular domain comprises residues 275 to 553 of SEQ ID NO:2, or anallelic variant thereof.
 6. An isolated polynucleotide according toclaim 1 which is a DNA as shown in SEQ ID NO:1 from nucleotide 339 tonucleotide
 1009. 7. An isolated polynucleotide according to claim 1wherein said polypeptide further comprises an affinity tag.
 8. Anisolated polynucleotide according to claim 7 wherein said affinity tagis polyhistidine, protein A, glutathione S transferase, substance P, oran immunoglobulin heavy chain constant region.
 9. An isolatedpolynucleotide according to claim 1 wherein said polynucleotide is DNA.10. An isolated polynucleotide encoding a polypeptide selected from agroup defined SEQ ID NO:2 consisting of residues 1 to 250, residues 1 to274, residues 1 to 553, residues 2 to 250, residues 2 to 274, residues 2to 553, residues 251 to 274, residues 251 to 553 and residues 275 to553.
 11. An expression vector comprising the following operably linkedelements: a transcription promoter; a DNA segment encoding aligand-binding receptor polypeptide, said polypeptide comprising asequence of amino acids selected from the group consisting of: (a)residues 30 to 250 of SEQ ID NO:2; (b) allelic variants of (a); and (c)sequences that are at least 80% identical to (a) or (b); and atranscription terminator.
 12. An expression vector according to claim 11wherein said polypeptide further comprises a signal sequence.
 13. Anexpression vector according to claim 11 wherein said polypeptide furthercomprises a transmembrane domain.
 14. An expression vector according toclaim 11 wherein said transmembrane domain comprises residues 251 to 274of SEQ ID NO:2, or an allelic variant thereof.
 15. An expression vectoraccording to claim 13 wherein said polypeptide further comprises anintracellular domain.
 16. An expression vector according to claim 15wherein said intracellular domain comprises residues 275 to 553 of SEQID NO:2, or an allelic variant thereof.
 17. An expression vectorcomprising the following operably linked elements: (a) a transcriptionpromoter; (b) a DNA segment encoding a chimeric polypeptide, whereinsaid chimeric polypeptide is comprised of a first portion and a secondportion joined by a peptide bond, said first portion consistingessentially of a ligand binding domain of a receptor polypeptideselected from the group consisting of: (i) a receptor polypeptide asshown in SEQ ID NO:2; (ii) allelic variants of SEQ ID NO:2; and (iii)receptor polypeptides that are at least 80% identical to (i) or (ii),and said second portion consisting essentially of an affinity tag; and(c) a transcription terminator.
 18. An expression vector according toclaim 17 wherein said affinity tag is an immunoglobulin F_(c)polypeptide.
 19. A transformed or transfected cell into which has beenintroduced an expression vector according to claim 11, wherein said cellexpresses a receptor polypeptide encoded by the DNA segment.
 20. Thecell of claim 19 wherein said cell is prokaryotic.
 21. The cell of claim19 wherein said cell is eukaryotic.